LTB4 is the most potent of all the arachidonic acid-derived mediators of neutrophil chemotaxis and degranulation. LTB4 also activates other leukocytes (eosinophils, lymphocytes, and monocytes) and in addition, exerts profound effects on vascular endothelial cells, pulmonary smooth muscle cells and bronchoepithelium, and skin. Ample medical evidence implicates LTB4-mediated activation of leukocytes and other cells in the pathogenesis of medical disorders as diverse as rheumatoid arthritis, ulcerative colitis, myocardial infraction, asthma, and psoriasis. All of these cell activation events appear to be mediated by the interaction of LTB4 with a specific cell surface molecule, the LTB4 "receptor". The LTB4 receptor remains unidentified. The identification and characterization of the LTB4 receptor is critical to the understanding of LTB4 signaling and the rational design of LTB4 inhibitors. The specific aims of this proposal are: Specific Aim 1) To obtain a cDNA clone for the LTB4 receptor. Two complementary approaches to clone the LTB4 receptor will be taken. a) Expression cloning in Xenopus oocytes. An assay for the mRNA encoding a functional LTB4 receptor has been developed using mRNA expression in Xenopus oocytes. Established screening strategies will utilize this assay to obtain the LTB4 receptor cDNA. b) Cloning by PCR and screening by expression in Xenopus oocytes. Considerable evidence suggests that the LTB4 receptor is a seven- transmembrane domain receptor. cDNA from LTB4- responsive cells will be subjected to PCR amplification using oligonucleotide primers based on consensus sequences of seven-transmembrane domain receptors and products will be used to screen cDNA libraries for full length clones. Pools of candidate clones will be screened for the ability to LTB4 responsiveness to microinjected oocytes, and if positive, will be progressively subdivided and rescreened until a single clone conferring LTB4 responsiveness can be identified. Specific Aim 2) To define the mechanism of LTB4 receptor activation and signaling. The LTB4 receptor cDNA clone will be used to address the following questions: Which structural domains in the LTB4 receptor mediate LTB4 binding? What is the relationship of the cloned receptor to the high and low affinity neutrophil LTB4 receptors and which neutrophil functions does it mediate? Where is the receptor expressed? Is the LTB4 receptor related to the receptors for leukotrienes C4, D4, and E4 or other arachidonic acid metabolites? Which G-proteins and second messenger pathways is the LTB4 receptor coupled to? Can structure-function studies with the LTB4 receptor clone be used to design receptor antagonists superior to the empirically-derived antagonists currently existing?